Spore cultivation



Sept. 19, 1961 s. SCHWEIGER 3000,791

SPORE CULTIVATION Fi1ed March 25, 1958 INVENTOR.

lwngzi5ahwa 977 3,000,791 SPORE 'CULTIVA'IION Leonard B. Schweiger, Elkhart, Ind., assignnr 10 Miies Laboratories, Inc.' Elkhart, Ind. a corpnration tat india.na

Filet! Max. 25, 19,58, Sex. N0. 723,996 3 Claims. (C1. 19536) This invention relates to the cultivation of Aspergillus niger spores.

More particularly it relates t the produc tion of Aspergillus niger spores Which are characterized by their improved propensity for increasing the yield and production rate of citric acid When used as the fermentative organism in the fermentation cf carbohyate-containing maten'als.

While it has been recognized, as for exarnple in U.S. Patent N0. 2492667, that the production ef citric acid by ferrnentation of carbohydrate-containing materials using Aspergillus niger is dependent upon the development of a proper cellular metabolisrn of the fermentative organism and that this metabolism is greatly influenced by the medium on which the spores have been cultivated, the problem of consistently obtaining economically attractiv e yields of Aspergz'llus niger spores having the right Kind and degree of physiological activity has up to t.l1e present time, not been satisfactorily solved.

Tims, though it is possible to obtain good physiologically activ e spores of Aspergz'llus niger by growing them on synthetic media, the spore yield is uneconomically meager. The use, as sporulating media, of non-synthetic material, such as wheat bran, pufied rice, polished rice and b ee t pulp, results in an abundance 015 spores which, however, have low activity insofar as citric acid production is concerned, Sinne of the other non-synthetic materials have presented a contamination problem Which made it extremely difiicult to carry out an aseptic sporepropagation proceclure.

In accorda.nce With the present process, it has been discovered that, corn bran, i.e., the produc-t represented by alle fraction of -corn which contains largely hulls or pericarp of corn kemel as obtained by dry milling, When wett ed with distilled water, sterilized, inoculated with a suspension of Aspergz'llus nz'ger spores grown 011 agar slants and aerated for a period of for eXam-ple, about seven to fourteen days resulted in t'ne pro-duction 0f crops of spores in. excellent yield with all the desired characteristics for citric acid fermentaticn. These spores, w hen used as the inopulum in a citric acid fermentation such as descrihed in U.S. Patent N0. 2492667 resulted in acid production rates and ylelds which were significantly better than those obtained with spores developed on other media.

My invention will be fully illustrated in the following described detailed procedure for the cultivation of Aspergillus m'ger spores, taken in conjunction with the drawing, in which there is illustrated a five gallon Pyrex carboy 11, fitted at; the mouth in known manner With an aluminum tube 12, serving as an exhaust air autlet. An air inlet tube 13, conveniently of glass 0a the like is attached to the aluminum tube 12, and projects into the carboy 11, about three-quarters of its dep-th, to prevent contamination. The exhaust air outlet tube 12 is covered With a conventional snap cap '14, which coutains a thick filter paper pad 14a to prevent contamination.

Exhaust air outlet tube 12, is maintained in close-fitting air-tight oonnection with the carboy 11, by meansof rubber gaskets 15 and a ring clamp 16, suitably of aluminum 0r the like, secured together by screws 17 as shown. A cotton air filter 18 connects the air inlet tube 13 and ai; flow meter 19 by suitable tubing. A rubber tu-be 21 oonnects the inlet tube 13 and the cotton air filter 18, and air flow may be shut oft by means of a pinch c1amp 19a on tube 21.

In use, twelve-hunclred grams 01 corn bran are placed in the five gallon Pyrex carboy 11, to which is added 1 liter cf distilled or otl1erwise deionized water, and the carboy shaken to moisten all the bran. While tl1e preoise amount of Wetter added at this point is not particularly critical, a large excess of water is undesirable since lt causes growth o-f the mycelium to such an extent that the bran may become matted, and ciots forma. This reduces spore production and increases diificulty in emptying carboy contents. Ami if an extremely small amount of Water Were used it may result in insuflicient spore produotion. About one liter per 1200 gmms of bran works quite well.

After the bran is moistened, the air exhaust tube 12 and the air inlet tube 13 are sealed through the use of. the snap cap 14 and the pinch clarnp 19a on tube 21, respectively. The carboy 11 is then placed in an auto-. clave and maintain ed there at 20 p.s.i. steam pressure for one and one-half to two hours. The carboy is then removed from the autoclave, cooled to somewhat below 50 C. and shaken well to break up all clumps of corn bran prior to the inoculation step Which follows.

Spores from 1 01 2 agar slants propagated 011 tl1e slants in accordance With the technique described in U.S. Patent N0. 2492667, are suspended in approximately 200 m1. of sterile distilled water containing a small amount 01 a wetting agent such as Tween er 81, to eff ect rapid and -thorough wetting 0f the bran and spores. Tween 80 is a trade name for sorbitan monooleate in the forrn 0f an oily solid, having a specific gravity of LOS-1.10; Tween 81 is a trade narne for sorbitan monooleate in the form cf an oily liquid, having a specific gravity 0f LOG-1.85. The entire suspensio-n is transferred to the carboy, care being taken to insure maintenance of strict aseptic conditions. The carboy may then be placed on a rack, the air line connected, and aeration maintained at a rate o-f 3 liters per minute. The carboys are rotated, for example, once a day, through the entire incubation period which may take from about seven to fourteen days to achieve maximum spare yield.

Spores Will begin to develop at approximately tw entyfour to thirty hours. During the first four or five days considerable heat is generated by the gmwing mycelium in the bran, although heat production will decre ase as the spare crop increases. A suflicient spare crop may develop in seven days, although it may -be advisable to continue the incubation for as much as seven additional days to obtain the maximum number of spores. During this time, the temperature of tl1e incuba-tion room, and Ehe air, is preferably held between about 28 C. and 30 C.

After appropriate incubation, the air line is disconnected at the carboy filter, approximately twelve 1iters 0f sterile distilled Water containing about 10 m1. of a we| fing agent such as Tween 80 01 81 are added to the carboy 11 through the air outlet tube 12, and the carboy is shaken vigorously to break up any large clumps of bran and mycelium, after which tne product is ready for addition to the fermenter.

The unexpectedly superior citric acidproducing proper ties of spores grown in accordance with rny invention were demonstrated by means of a side-by-side compa1ison of performance characteristics of such spores with spores grown 0n other media. In these comparative tests, fermentations were conducted according to the procedure given in U.S. Patent No 2476159. Cellular morphology used as a convem'ent means cf control, was determined by macroand microscopic examination, and citric acid was calculated as the monohydrate, by titration with 0.1 N sodium hydroxide.

The inoculum used in these comparative tests cousisted f spore suspensions of Aspergillus m'ger produced on corn bran, wheat bran, rye grains, Sabourauds agar slants, polished rice, beet pulp, and the prepared dry breakfast cereals sold under the trade names cf Puffed Wheat, Kix, and Cheerios. The spores were separated from the sporulation medium through the use of sieves 011 a shaker, except that spores Which were developed on corn bran and wheat bran were not separated from the bran, but used as a spore-bran suspension. The amounts of spores used were based upon a standard prepared by washing the spores oft two agar slants and diluting tl1e suspension t0 100 m1. Five m1. of this suspension was lised as a standard inoculum for 4000 ml. of media. Except for spores produced on corn bran and wheat bran, spore suspensions were made up to the same density as the standard inoculum. Corn bran plus spores and Wheat bran plus spores were each made up into suspensions Which contained 50 Ing. of solids per rnl, The standard was approximated by using 1.5 ml. cf the spore-bran suspension per 4000 m1. of the media Table I below shows the comparative eifectiveness of a number of substrates with respect to size of resulting Spore crop, ease of sterilization cf Spore crop, cost and availability of substrate, and physiological (citric-acid producing) activity.

TABLE I Same general observations 0f sporulmion substrates Sporulation (lost and Physio Substrate Spore Orop Sterilizat-ion Availability logical Act-ivlty Sabouraud's Moderate. Oomplete... High cost avail- Very good.

agar. able. Rye graln Excellent- Lengthy Low cost readily D0.

and difl1- available. cult. Beet pulp-.-- Good----- Complete.-- Low oost avail- Very poor.

8. G. Ries ---do .do--.----.- Poet. T(ix Higbl cost. avail- Very poor.

a e. Cheerlos dn (in 'Do. Puffed -.do du Erratic.

Wheat. Wheat Bran.. Excellent- Lowcost readily D0 available. Garn bran--. ---do ..do Very good.

With respect to Table I, it is pointed out that while Sabourauds agar is generally a goocl substrate for laboratory tests where quantitative duplication of results are important, its use as a sporulation medium for large scale production is generally prohibitive because of the high Cost of the agar, and the time and labor which is required f0r harvesting the spare crops.

Rye grain is cheap, readily available and was found to produce especially good crops of spores. However, the spore harvest procedure is somewhat diificult in that the spores must be separated frorn the grain prior to preparing a suspension for inoculati0n. The further disadvantage lies in the fact that the time and temperature required for sterilization also chars the grain which then results in an uneconornical crop of spores.

Beet pu1p, rice, Kix, Cheerios, Pufied Wheat and wheat bran likewise did not meet the necessary practical requirements cf a commercially satisfactory substrate either brecause of high cost, poor er erratic physiological activity or both.

Corn bran, 011 the other band, met all of the requirements in that luxuriant spore crops were produced that developed into high acid-producing mycelium. Corn bran is cheap, readily available, can be sterilized rapidly and completely, and the spores do not have to be separated from the bran prior to the inoculation of a fermentation.

In determining the citric acid-producing activity of mycelia produced from spores from the various sporulation media listed in Table I tl1ose spoms Were ed aS Evaluation 03 A. niger spare inocula developed an various sporulalz'on media as determined by citrz'c acid production Total Sporulation media Fermentation media. acidity Sabourauds agar Corn sugar. 359 Ry@ gram Gern Sugm- 333 Gern brzm Beet pulp--. 0 Rice Kix (cereal) 134 Oheerios (cereal) 0 Pufied Wheat (cereal) 8 Wheat Bran 0 Of the nine sporulation media studied it is seen that only Sabourauds agar, rye grain, and corn bran, procluced spores that germinated into typical mycelia w-ith the proper cellular metabolism for acid production in the Substrates employed.

Ritze and Kix pr0duced spores that developed into a borderline type of growth that produced citric acid in quantities far inferior to the Sabourauds agar.

Beet pulp, wheat bran and the cereals, Cheerios and Pufied Wheat produced spores that germinated into mycelia with the improper cellular metabolism for producing citric acid.

As a further evaluation of the spores developed on c'orn bran, a comparison was made With spores developed 011 Sabourauds agar and spores developed cm rye grain, in regard to their ability to produce citric acid from three difierent substrates:

(1) Decationized N0. 70 00m sugar; (2) Decationized high test molasses; (3) Refined cane sugar.

The citric acid yields from the various substrates using spores produced 0n the three sporulation media listed above are shown in Table III below:

TABLE 111 A comparison 0] inocula grown 071 Sizbouraud's agar rye grain and c0rn bran as determined by cizric acid production from #70 com sugar, high test molasses and sucrose substrates Converslon of Initial earbohydrate to citrlc acid (percent) Inoculum Fermentation substrate 1 Average conversion. Figures in parenthesis indicate number of iermentations included in average.

The superiority of i1 1ocula preparedfrom speres developed on corn bran over spores produced 011 rye grain and Sabourauds agar is clearly shown by a comparison of acid yields obtained irom all three substrates.

It was also found that the corn bran spore fermentatins were able to tolerate a higher initial sugar content because of more rapid acid production rates, which allowed more sugar to be fermented out of the saure fermentation as the rye grain spore fermentations With lower initial sugar levels. It has been found further that the eificiency of citric acid production in fermentations using decationized high test molasses is greatly improved with the use of spore in0cula developed on corn bran. Ami additionally, since spores do not have to be separated frorn the bran, the present technique permits the whole spore development and inoculation procedure to be carried out under the strictest. aseptic couditions, which is highly advantageous.

In summary, the present technique for producing spores of Aspergillus m'ger permits production of a surprisingly great abnndance of spores under aseptic couditions. Inoculation of fermentation media with spores produced in accordance with my new technique not only does not require separation of spores from bran, thereby permitting introduction of bran and spores into the fermentation medium together, but also produces a mycelial growth having unexpectedly high citric acid-producing properties.

I claim:

1. A method of preparing inocula for use in the submerged fermentation of citric acid which comprises References Cited in the file of this patent UNITED STATES PATENTS 1,564385 Waksman Dec. 8, 1925 2,047,669 Cahn July 14, 1936 2,278236 Ayres Mar. 31, 1942 2352168 Christensen Inne 27, 1944 2,385,283 Karre et a1 Sept. 18, 1945 2476,159 Schweiger et a1. July 12, 1949 FOREIGN PATENTS 348,214 Great Britain May 14, 1931 OTHER REFERENCES Thom et al.: Manual of the Aspergilli, 1955, published by The Williams & Wilkins Co. Baltimore, Md., pp. 37 and 38.

Lilly et al.: "Physiology of the Fungi, 1951, publishecl by McGraw-Hill Book Co., Inc., New York, pp. 333 and 334. 

1. A METHOD OF PREPARING INOCULA FOR USE IN THE SUBMERGED FERMENTATION OF CITRIC ACID WHICH COMPRISES INOCULATING STERILIZED CORN BRAN WITH SPORES OF ASPERGILLUS NIGER IN THE PRESENCE OF SUFFICIENT STERILE WATER TO WET THE BRAN, AND INCUBATING THE RESULTING MIXTURE IN THE PRESENCE OF AIR UNTIL A SUBSTANTIAL CROP OF THE SPORES HAS BEEN PRODUCED. 